The goal of the proposed research is to investigate neuron and muscle excitability in the nematode C. elegans. We have identified over 30 genes that regulate excitation of defecation, egg-laying, and body-wall muscles, and we have molecularly cloned five of these genes. The genetics of four of these is strikingly similar: each has dominant gain-of-function mutations (gf) that cause strong defects in muscle excitation, and loss-of-function (lf) mutations cause little or no obvious phenotype. All four genes encode K+ channels. We think that the gf mutations in each K+ channel cause channel activation in vivo, accounting for their strong excitation defects. The fact that lf mutations cause relatively minor defects suggests that the many K+ channels have overlapping functions in vivo. We will continue analysis of these genes and other similar genes identified in genetic screens. The K+ channels will be expressed in cultured cells to study their electrophysiological properties. It will be particularly interesting to study how the gf mutations affect channel properties. Two of these K+ channels are related to the human HERG channel, defects in which cause a cardiac malfunction called long-QT. Long-QT can also be caused by tricyclic antidepressants and certain cardiac anti-arrhythmic drugs. We have evidence that these drugs also block one (but not the other) of the C. elegans HERG-related channels in vivo and in vitro. We will study the C. elegans channels and their human equivalents to understand the basis for this specificity and its implications for long-QT disorder. We have shown that the fifth muscle excitation gene, called unc- 43, encodes the nematode homologue of calcium-calmodulin dependent protein kinase II (CaMKII). CaMKII is implicated as a key regulator of synaptic activity, particularly of synaptic plasticity that underlies learning and memory. We have many lf mutations in unc-43, including null mutations. These mutants are viable and have complex behavioral abnormalities. There is also one gf mutation in unc-43, and we think that this mutant CaMKII is partially Ca++ independent (activated). This gf mutant is also viable and confers complex defects that are the opposite of those in null mutants. We have begun to use the unc-43 activated mutation to identify extragenic suppressors of its various phenotypes, some of which we expect to encode direct CaMKII substrates. We propose to use a combination of genetic analysis, molecular cloning, and biochemical analysis to characterize these potential targets and to determine whether they are directly phosphorylated by the unc-43 CaMKII. We will also continue genetic screens to identify additional potential targets.